Secretome of adipose derived stem cells induced apoptosis in anaplastic thyroid carcinoma C-643 cells

1Department of Basic Sciences, School of Medicine, Bam University of Medical Sciences, Bam, Iran 2Cellular & Molecular Research Center, Medical Basic Sciences Research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran 3Clinical Microbiology Research Center, Ilam University of Medical Scienes, Ilam, Iran 4Department of Virology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran Immunopathologia Persa http www.immunopathol.com

ATC is one of the thyroid malignancies that is very aggressive, has a high mortality and is a threat to human health. Unfortunately, the probability of living more than five years after contracting this disease is less than 5% and most patients die in the early months after being diagnosed. ATC is more common in older people (with an average age of about 60) and is more common in women than in men (3,4).
The probability of successful surgery depends on many factors such as the age of the person, the presence of the underlying disease and the rate of progression of the disease at the time of diagnosis.
Chemotherapy and radiotherapy procedures can also damage normal cells (5,6). Thus, it is necessary to discover a new treatment method, which is not harmful to normal cells. several sources, such as bone marrow, adipose tissue and umbilical cord. They are multipotent cells that have self-renewal and expansion potential. Additionally, they secrete many paracrine factors, which have anti-inflammatory and anti-cancer properties and are called the secretome (7)(8)(9). Due to the secretion of paracrine substances, MSCs have a great effect on the treatment of diseases, including various cancers.

Mesenchymal stem cells (MSCs) have
MSCs play a dual role in modulation of cancer cells and can either inhibit or support the growth of tumor cells. Previous studies have shown that MSCs can affect a variety DOI:10.34172/ipp.2022.20 2 of cancers. However, the results of these studies are contradictory and the precise mechanism of this effect has not yet been determined (12)(13)(14)(15).
The results of these studies depend on numerous factors such as origin and type of tumor, type of study and animal species (16). Identifying the effect of the scrotum on the signaling pathways leading to cancer cell death can help treat various cancers.
Among the MSC sources, adipose tissue is more abundant and accessible (17,18). Adipose-derived mesenchymal stem cells (ADMSCs) possess to release ability of cytokines, hormones and growth factors. In this study, the effects of ADSCs secretome on an ATC cell line (C-643) were evaluated.

Objectives
Given the many benefits of MSCs, this study aims to discover a new method for treating thyroid cancer that could be used to make anticancer drugs.

ADSCs isolation and characterization
Human subcutaneous adipose tissue was achieved through liposuction surgery and transferred to the cell culture laboratory under sterile conditions. After washing with phosphate-buffered saline (PBS), the fat pieces were exposed to type I collagenase (1.0 mg/mL in DMEM) for 30 minutes at 37˚C. Then, the obtained homogenous cell suspension was centrifuged at 1200 rpm for 10 minutes. The obtained cell pellet was re-suspended in DMEM and then cultured in 25 cm 2 flasks. The culture medium was exchanged every three days and when the confluency gained about 80%, the cells were passaged. After 3 rd passage, cell characterization was conducted using flow cytometry to determine specific markers. Alizarin-red and oil red O staining was used to evaluation of osteogenic and adipogenic differentiation (Figure 1).

Culture of C-643 cells
ATC cell line (C-643) was purchased from National Center for Genetic Resources of Iran. C-643 cells were grown in RPMI 1640 medium supplemented with 1% penicillin-streptomycin, 2mM L-glutamine and 10% fetal bovine serum and were incubated in the presence of moist temperature with 5% CO 2 at 37˚C ( Figure 2).

Preparation of secretome
The ADSCs (after 3 rd passage) were cultured in media supplemented 10 % FBS until 80 % confluence. The cells were washed with PBS, and incubated with serumfree media for 20 hours. The conditioned medium was collected and centrifuged for 10 minutes (4°C, 4000 g). Then, 10 mL conditioned medium re-centrifuged (4000 g) with Amicon Ultra-15 centrifugal filter devices with 3000 molecular weight cut off membrane (Millipore, USA) at 4°C for 2 hours. The BCA kit (Thermo Scientific, USA) was utilized to assess the protein content of the supernatants in accordance with the manufacturer's protocol, stored at -80°C until use.

Cell viability assay
Cell viability was assessed by MTT assay for evaluation of cytotoxic effect of ADMSCs secretome on C-643 cells.
Briefly, culturing C-643 (1 × 10 4 cells/well) cells was conducted in 96-well plates. After 24 hours, the culture medium was replaced with various concentrations (25 μg and 50 μg) of secretome for 24 and 48 hours. When the cells were treated, MTT solution with 0.5 mg/mL concentration was added to wells, then the plate was incubated at 37°C for 4 hours. Then the supernatant was drained and 100 µL of DMSO was added to all of the wells. Finally, the absorbance was measured at 570 nm by a microplate reader (Bio-Rad, CA, USA). IC50 values were determined using MTT assay. Each trial was conducted by triplicate.
Colony assay C-643 cells were seeded on 6-well plates at a density of 500 cells per well. The cells received complete media supplemented with 25-μg and 50-μg secretome. After 14 days, the cells were stained with 0.1 % crystal violet colored solution for 15 minutes. Finally, the plates were washed and air-dried and a colony count was conducted.

Acridine Orange/Ethidium Bromide (AO/EB) apoptosis assay
AO/EB staining was utilized to distinguish apoptotic cells. Briefly, C-643 cells were cultured in 96-well plate (4×10 5 cells/well) and incubated for 24 hours. The cells were treated with 25μg and 50-μg secretome for 24 hours. Following cells washing with PBS, 20 μL of cell suspension was mixed with 1 μL of AO-EB working solution (1 μL of AO and EB stocks plus 1 mL PBS) and the cells were looked under fluorescence microscope immediately. The green and orange cells represented living and dead cells, respectively.

Real-time polymerase chain reaction (RT-PCR)
Total RNA extraction was conducted using the RNeasy Mini kit (QIAGEN) In accordance with the manufacturer's instructions. RNA concentration and purity were measured using Nanodrop spectrophotometer. cDNA was synthesized using a cDNA Synthesis kit from 6 µg of total RNA regarding the manufacturer's instruction. About 2 μL of cDNA was augmented in each 25 μL PCR reaction compound with 12.5 μL of 2x SYBR Green Master Mix (Fermentas, Canada), 10.1 μL DEPC water, reverse primers, and 0.2 μL of each 10 pM forward. Table  1 reports sequences for each primer. PCR was amplified in 40 cycles via the program below: 95°C for 10 min, 95°C for 15 seconds, and 95°C for 30 seconds, and also 60°C for 34 seconds. Data analysis was conducted by the 2 -ΔΔCT method. Expression values were corrected for the housekeeping gene GAPDH

Statistical analysis
Data has been analyzed by SPSS 21.0 (Chicago, IL, USA) through ANOVA. Then, post hoc pair-wise comparison was conducted by Bonferroni technique. P value ˂ 0.05 was regarded as statistically significant. Each trial was conducted in quadruplicate.

Cell viability
After 24 hours incubation, no significant difference was seen in the viability percentage of 25 μg/mL treated cells compared with control group. The viability percentage of 50 μg/mL treated cells after 24 hours incubation was remarkably diminished compared with the control group (P < 0.01). After 48 hours incubation, the viability percentage of cells was considerably diminished from 100% in control to 48 and 29% in 25 μg/mL and 50 μg/mL treated cells. Additionally, in 50 μg/mL treated cells, the survival rate of cells was remarkably more than 25 μg/mL (P < 0.05) (Figure 2).

Colony assay
In 25 μg/mL treated cells, the colony numbers were remarkably lower than control group (P < 0.01). The colony formation in 50 μg/mL treated cells was remarkably diminished compared with control group (P < 0.001) and 25 μg/mL treated cells (P < 0.05; Figure 3).

AO/EB apoptosis assay
AO/EB double staining protocol is a quick and reliable fluorescence technique to differentiate the viable, apoptotic and necrotic cells. The nuclei of living cells appear bright green. The nucleus of apoptotic cells emits an orange color. Red nuclei indicate necrotic cells (19).
Around 94% of cells in the control group were viable, 4% were in apoptosis and 2% were in necrosis stage. The percentage of viable cells was diminished to 66% in 25 μg/ mL and 40% in 50 μg/mL secretome (se)-treated cells. In 25 μg/mL se-treated cells, the apoptosis percentage was significantly more than the control group (6.25-fold, P < 0.01). The apoptosis percentage of 50 μg/mL se-treated cells was noticeably more than control group (11.75-fold, P < 0.001) and 25 μg/mL se-treated cells (1.88-fold, P < 0.05).

Gene expression assessment
In 25 μg/mL se-treated cells, the expression of Bax gene was significantly more than control group (2.61-fold, P < 0.01). The expression of this gene in 50 μg/mL se-treated cells was remarkably more than control group (3.37-fold, P < 0.001) and 25 μg/mL se-treated cells (1.4-fold, P < 0.05).
In 50 μg/mL se-treated cells, the expression of Bcl-2 was lower than control group and 25 μg/mL se-treated cells (P < 0.05).
The expression of P53 gene in 50 μg/mL se-treated cells was significantly more than control group and 25 μg/mL se-treated cells (P < 0.05; Figure 5).

Discussion
Previous studies have proven that MSCs have many considerable regenerative attributes, because they can produce a variety of soluble factors that affect tissue structure.
The results of this study showed that exposure of C-643 cells to ADSCs-secretome caused the reduction in viability and cell proliferation while the apoptosis was enhanced. These changes were dose-dependent, so that the maximum was seen in 50 μg/mL se-treated cells. In agreement with this study, Maj et al introduced that conditioned medium of ADMSCs could diminish the viability and proliferation of bladder cancer cells (20). Contrary to this research, Onzi et al, concluded that conditioned medium of ADMSCs had no effect on proliferation of human glioblastoma cancer stem cell but could alter glioblastoma cells migration (21).
In the study by Bielli et al, it was found that in the tumor microenvironment it seems that ADSCs-secretome influence active breast cancer cells and increase neoangiogenesis, matrix remodeling and intercellular communication through Gap Junction, thereby promote cancer metastasis (22).
Apoptosis is one of the most important mechanisms utilized to study the therapeutic effects of anti-cancer drugs. AO/EB assay can easily identify viable, apoptotic and normal cells.
The study of Liu et al showed that dual EB/AO staining is an economic and appropriate method to identify apoptosis in tumor cells compared to flow cytometry. When AO penetrated into normal and early apoptotic cells, it bound to DNA and emitting fluorescent green light. EB interfered to late apoptotic and necrotic cells with damaged membranes bound to DNA fragments or apoptotic bodies and emitting orange-red fluorescence (23).
Exposure of C-643 cells to ADSCs-secretome caused the dose-dependent enhancement of apoptosis and necrosis mechanism.
RT-PCR was another mechanism was utilized to confirm the occurrence of apoptosis in the treated groups. For this purpose, the amount of mRNA expression of Bax, Bcl-2, p53, caspase-3 and -8 was evaluated.
P53 is a key gene in regulating of cell cycle and tumor suppression. In normal conditions, p53 exits in low levels. However, its expression highly induces under various stresses such as DNA damage and oncogenic activity that leads to form the net of signaling pathways. One of prominent outcomes of p53 activation is apoptosis inducing that can initiate through several pathways. In fact, P53 function as transcriptional factor can activates the expression of a large number of genes including Bax, Figure 5. ADSCs-Se effects on gene expression. Expression is shown as fold change relative to control untreated cells. Each assay has been accomplished 5 times, and the mean ± SD have been presented. *P < 0.05, **P < 0.01, #P < 0.05; * and # symbols indicate comparison to the control and 25 μg/ mL ADSCs-Se groups, respectively. Bak, Puma, Noxa and Bid as well as death receptors which promote apoptosis. On the other hand, the increase of the expression of these genes leads to increase the ratio of pro to anti-apoptotic proteins and consequently, induce extrinsic and intrinsic apoptosis by caspases activation. Bax and Bcl-2 genes are involved in setting the intrinsic pathway of apoptosis. They are the main members of the Bcl-2 family (24)(25)(26).
Exposure to ADSCs-secretome remarkably increases the expression of Bax gene, whereas the Bcl-2 expression was considerably diminished. Chiu et al have demonstrated that condition medium of ADSCs causes the increase of Bax, Caspase-3 and -9 gene expression, while the expression level of Bcl-2 gene was decreased (27).
The study of Yu et al revealed that condition medium of ADSCs causes the significant increase of Bax protein and reduction of Bcl-2 protein synthesis in bladder tumor cells (28).
In the present study, the expression of Bax, P53, caspase-3 and -8 was significantly increased in ADSCs-secretome treated cells, while Bcl-2 expression was decreased.
Pires et al, demonstrated that cytokines released by bone marrow derived MSCs, such as tissue inhibitor of metalloproteinases-1, and cytokine-induced neutrophil chemoattractant-1 induces differentiation and neurite outgrowth in SH-SY5Y cells (29).
High levels of caspase-3 and -7gene expression caused the onset of intrinsic apoptosis pathway in the cell (29). The study of Tummers et al revealed that the secretion of TNFα-related apoptosis-inducing ligand by MSCs begins a signaling cascade of caspase-8-dependent apoptosis and activates the extrinsic pathway of apoptosis (31).

Conclusion
The results of this study revealed that ADSCs-secretome had toxic effects on ATC (C-643) cells in vitro. The findings also indicated that ADSCs-secretome could inhibit the progress of thyroid anaplastic carcinoma through the activation of intrinsic and extrinsic pathways of apoptosis . ADSCs-Se effects on Caspases activity. Each assay has been accomplished 4 times, and the mean ± SD have been presented. *P < 0.05, **P < 0.01, P < 0.001; #P < 0.05; * and #symbols indicate comparison to the control and 25 μg/mL ADSCs-Se groups, respectively.
in cancer cells. Confirmation of this claim requires further research in this area both in vitro and in vivo.

Limitations of the study
Due to financial problems, we were not able to perform more laboratory techniques. Of the different types of thyroid cancer cell lines, we could only study the C-643 cell line.
Authors' contribution FRT participated in data collection, analysis and manuscript writing. AS was responsible for supervision and protocol development. SA was included in preparing the concept and design. SKH and SHM assisted in data collection. All authors participated in preparing the final draft of the manuscript, revised the manuscript and critically evaluated the intellectual contents. All authors read and approved the content of the manuscript and confirmed the accuracy or integrity of any part of the work.

Conflicts of interest
The authors declare that they have no competing interests.

Ethical issues
The Ethics Committee of Bam University of Medical Sciences approved this study (Ethical code # IR.MUBAM.REC.1400.005). The research also followed the tenets of the Declaration of Helsinki. This study was extracted from a research project was conducted in Bam University of Medical Sciences. Additionally, ethical issues (including plagiarism, data fabrication and double publication) have been completely observed by the authors.